Th17 cells are a subset of T helper cells which have been associated with several chronic inflammatory and autoimmune diseases. Upon polarization and activation Th17 cells produce highly proinflammatory molecules like cytokines IL-17A, IL-17F, IL-21, IL-22 and chemokines CXCL8 and CCL20. Furthermore, Th17 cells produce effector molecules like GM-CSF as well as provide help to B cells, induce germinal center formation and class switching.
The effector mechanisms and exacerbating Th17 responses are associated with the pathogenesis of acute and chronic inflammatory diseases like psoriasis, rheumatoid arthritis (RA), multiple sclerosis (MS), systemic lupus erythematosus (SLE), diverse phenotypes of inflammatory bowel diseases (IBD), asthma and allergy.
In rheumatoid arthritis the role of Th17 has been confirmed in several mouse models as well as in patient's material. High frequency of IL17 producing cells has been found in diseased synovium, and intra-articular administration of IL17 in collagen-induced arthritis models has led to worsening of RA symptoms. Furthermore, IL17RA deficient mice develop only very mild forms of RA. To date, anti-IL17A and anti-IL17RA monoclonal antibodies have been shown protective from progressive RA in humans and are approved as therapeutics.
In psoriasis, psoriatic arthritis as well as plaque psoriasis, chronic activation of Th17 cells and pro-inflammatory response against skin autoantigens as well as skin microbiota is the major cause of pathology. Respective patients' skin biopsies show high levels of IL17, IL23, IL6 and IL12, which represent the proinflammatory mixed Th1/Th17 phenotype. Additionally, Th17 cells in psoriatic plaques produce CCL20 and recruit more Th17, mast cells & neutrophils to prolong and elevate the response. Further, Th17 plays a role in inflammation processes that are part of several other skin phenotypes like acne, dermatomyositis and scleroderma.
Beside the skin diseases, Th17 cells play also an important role in further mucocutaneous inflammatory diseases such as chronic, allergic and steroid-resistant asthma, chronic obstructive pulmonary disease (COPD) and inflammatory bowel diseases, like Crohn's disease, enteritis and ulcerative colitis. IL17A has been found in high levels in sputum of severe asthma patients as well as of COPD patients. IL17 is potently induced via aryl-hydrocarbon receptor (AhR), a target of cigarette smoke ingredients and several pollutants.
The pathology of IBD is an intensively studied field and convincing data, despite diversity of models used, show the importance of Th17 cells. In Crohn's disease patients experience reoccurring disease phases with increased Th17 frequencies. The strong Th17-driven autoimmune response might be linked to the IL23R polymorphism, and the overshooting host defense against gut microbiota is based on a multiplicity of Th17-mediated mechanisms. Nevertheless, the inhibition of IL17 was not shown beneficial in Crohn's disease, which mirrors the diversity of IBD phenotypes in the patient population.
One of the first diseases that was shown to be Th17-driven was multiple sclerosis and although the complexity of the disease increases, Th17 cells still remain in the central role. Diverse cell types from the brain biopsies of MS patients showed IL17A overexpression and extensive infiltration of lymphocytes. Furthermore, the peripheral phenotype of MS seems also to be Th17-dependent, as these cells have been shown to provide B cell help and may induce autoantibody production.
As in MS, similar involvement of Th17 cells has been shown in another autoimmune phenotype—systemic lupus erythematosus (“SLE”). This complex and diverse disease worsens with increase of Th17 cell numbers. Major effect of Th17 in SLE was observed in progressive vasculitis and endothelial dysfunction. This effect is not only characteristic to SLE, but also to Behcet's disease, uveitis and Sjögren syndrome. IL17-producing cells have been found to accumulate in vessel walls of autoimmune vasculitis patients and recruit other proinflammatory cells, like neutrophils and macrophages. IL17 has prothrombic and procoaggulant effects on human endothelial cells.
Concomitantly with accumulation of Th17 in vasculitis there is a positive correlation between Th17 frequency and frequency of aortic lesions in coronary atherosclerosis. Here, increase in Th17 levels lead to increase in size of aortic lesions and frequency of cardiac infarction.
Beside rheumatoid arthritis, Th17 plays a role in another chronic inflammatory joint and spine disease, Morbus Bechterew. Here, during ongoing tendon inflammation Th17 and mast cells both produce IL17 in the synovium and drive ectopic bone formation and joint stiffness. The ongoing inflammation is fairly treatable with anti-TNF alpha therapy but the bone reformation and ankyloses might be Th17 dependent.
One yet partially explored field of Th17 cells is neuropathic pain. There is accumulating data that diverse neuropathic and chronic unspecific low back pain correlate with the disrupted balance between Th17 and regulatory T cells.
Taken together, inhibition of Th17 development and function appears to clearly be a promising target for the treatment of the above-mentioned diseases.
The master transcription factor of Th17 cells is ROR gamma t. Retinoic acid receptor-related orphan receptors (RORs) are members of the steroid hormone nuclear receptor family. The ROR transcription factor subfamily consists of three members: ROR alpha (RORA), ROR beta (RORB) and ROR gamma (RORC). Each member is expressed as independent gene and binds as monomer to genomic response elements. Through alternative splicing and differential promoter usage each ROR member generates variants that have different tissue expression patterns and that regulate different targets. RORC has two isoforms that arise from differential promoter usage and that differ in the first exon. These two transcript variants are annotated as: NM_005060 for ROR gamma (RORC1) and NM_001001523 for ROR gamma t (RORC2). While ROR gamma is expressed in a variety of tissues either constitutively or under circadian rhythm, ROR gamma t expression is limited to the cells of the immune system. The highest expression of ROR gamma was shown for skeletal muscle, kidney and liver while ROR gamma t expression peaks in the developing double positive thymocytes, T helper lineage 17 cells (Th17), lymphoid-tissue inducer cells (LTi) and several intraepithelial lymphoid cell (ILCs) subpopulations. ROR gamma t has a critical role in thymopoiesis as it reduces Fas expression and IL2 dependency, rescuing the thymocytes from the activation-induced cell death during thymic selection. Further, ROR gamma t is important for the function of LTis and development of secondary lymphoid tissues.
Thus, inhibition of Th17 development and function through the inhibition of the master differentiation factor, ROR gamma t, appears to be a viable option.
One possible approach for inhibiting the Th17-driven pathologies is to inhibit the effector functions of already present Th17 as well as interfere with the new differentiation of nave T cells towards Th17 lineage by blocking ROR gamma t. However, ROR gamma t is an intracellular molecule and cannot be reached by therapeutic antibodies. Furthermore, the currently available ROR gamma and gamma t small molecule antagonists target the ligand or DNA binding domains that are identical between the both molecules. As previously stated, ROR gamma is ubiquitously expressed and has roles in many central physiological processes. The most studied role of ROR gamma is the circadian regulation of lipid and sugar metabolism in liver and skeletal muscle. The absence of ROR gamma leads to imbalance of liver metabolic function, metabolic syndrome, insulin resistance and obesity. Additionally, several studies have evidenced an important role of ROR gamma in cancer. ROR gamma deficiency in mice leads to a high incidence of thymic lymphomas with frequent liver and spleen metastasis. Thus, there is a high an unmet need for ROR gamma t-specific inhibitors.
One alternative approach for inhibiting the Th17-driven pathologies is to knock down the expression of ROR gamma t on the nucleic acid level. Such targeting has several important advantages, since in comparison with the available small molecule therapeutics, targeting on the nucleic acid level may be adjusted to target the ROR gamma t transcript exclusively, thus having no effects on ROR gamma. Small molecules currently in development do not have this level of specificity which may lead to adverse side-effects with these drug candidates. However, the initial approach of using DNAzymes based on the 10-23 motif, which had already been successfully applied to the inhibition of GATA-3 (see WO 2005/033314) did not result in an inhibition of the expression of RORC2 relative to untreated control in HDLM2 cells by at least 50% (see Example 2).
WO 2006/007486 is based on examining the influence of RORC2 expression on the proliferation of immune cells and claims the use of inhibitors or antagonists of RORC2 expression. Antisense constructs are listed as potential inhibitors, but no working examples are shown.
WO 2012/129394 relates to the treatment of cancer by using IL-9-receptor agonists wherein the agonists can act either directly on the receptor or indirectly by regulating RORC2 expression. Antisense constructs are listed as potential inhibitors, but no working examples are shown.
CN 102 732 562 apparently describes siRNA-based inhibition of, for example, RORC1, including siRNA constructs according to SEQ ID NOs: 7 bis 9. No siRNA constructs against RORC2 appear to be shown.
WO 2011/113015 relates to the use of anti-sense constructs against nuclear hormone receptors (“NHRs”). While RORC1 is listed as an example of such NHRs, RORC2 is not listed.
Hakemi et al.; Avicenna Journal of Medical Biotechnology 5 (2013) 10-19, relates to the siRNA-based gene silencing of RORC2, showing in total three anti-RORC2 siRNA constructs (see Table 3). These constructs, however, do not appear to be RORC2-specific since they correspond to regions of RORC2 that are shared with RORC1 (start positions 872, 1197, and 1303, respectively, of SEQ ID NO: 1 shown herein in FIG. 1).
Burgler et al., J. Immunol. 184 (2010) 6161-6169 examines the interaction of RORC2 with the FOXP3 promotor, including the use of two siRNA constructs in the knock-down of RORC2 (see Supplemental table 4). These constructs, however, do not appear to be RORC2-specific since they correspond to regions of RORC2 that are shared with RORC1 (start positions 875 and 2473, respectively, of SEQ ID NO: 1 shown herein in FIG. 1).
Webering “Zur Rolle des α-Melanozyten-stimulierenden Hormons (α-MSH) and des retinoid-related orphan receptor γt (RORγt) bei der Immunpathogenese des Asthma bronchiale”, PhD thesis, Lübeck 2014; Kategorie “Y”) relates to the role of RORC2 in the immune pathogenesis of asthma bronchiale, including the use of DNAzymes and of three different siRNA constructs for inhibiting RORγt expression. While the use of DNAzymes could not be shown to have any influence on RORγt expression (see Section 3.2.3 and FIG. 36), siRNA constructs apparently resulted in a reduction of RORγt expression by up to 65% after 48 h (see Section 3.2.4 and FIG. 38). However, it is unclear which regions of RORγt were targeted, since the sequences shown in Table 5 do not match with the RORC2 sequence shown as SEQ ID NO: 1 herein in FIG. 1.
WO 2004/024879 relates to the interaction of RORs with the p21 pathway. In total, DNA sequences are shown for 15 different RORs, apparently including RORC2 (SEQ ID NO: 12). For three of those RORs, but not for RORC2, siRNA-based knock-down experiments are shown.
Lin et al., Mediators Inflamm. 2015: 290657 and Song et al., Biochem. Biophys. Res. Commun. 452 (2014) 1040-1045, relate to the inhibition of RORC2 with small molecules and aptamer-siRNA-chimerae. No sequence information is apparently given.
Thus, therapeutic agents which are able to specifically inhibit the expression of ROR gamma t intracellularly are essential for the treatment of Th17-driven diseases, in particular inflammatory disorders of various organs. Hence, there is still a high scientific and medical need for therapeutic agents, which reduce or inhibit RORC2 expression and/or activity. Particularly, there is a longstanding need for oligonucleotides, which specifically interact and thus, reduce or inhibit the expression of RORC2, as well as oligonucleotides, which specifically inhibit RORC2, without causing any (severe) side effects.